The present invention relates to primers for the detection of genes for drug metabolizing enzymes, especially for the detection of the presence or absence of mutated nucleotide sequences within the genes of "poor metabolizers" (PMs) of drugs. The invention further relates to a method and a diagnostic kit for the detection of such genes or nucleotide sequences using Polymerase Chain Reaction (PCR)-technology.
Polymorphic genes play an important role as causes of interindividual variation in drug metabolism and in the occurrence of side effects and therapeutic failures. Moreover, they serve as genetic markers for numerous diseases. The elucidation of these mutations therefore has clinical importance and routine phenotyping has been recommended particularly for psychiatric patients and for volunteers in clinical studies (Gram and Br.o slashed.sen, European Consensus Conference on Pharmacogenetics. Commission of the European Communities, Luxembourg, 1990, pp. 87-96; Balant et al., Eur. J. Clin. Pharmacol. 36, 551-554 [1989]). Moreover, recent studies have indicated that a link may exist between the debrisoquine phenotype and some forms of cancer (Caporaso et al., Cancer Research 49, 3675-3679 [1989]).
The existence of the polymorphic oxidation of debrisoquine and sparteine reported by Mahgoub et al., Lancet 1977, pages 584-586 and Eichelbaum et al., Eur. J. Clin. Pharmacol. 16, 183-187 [1979], caused a resurgence of interest in genetic factors influencing the individual response to drugs. Today, the debrisoquine polymorphism probably is one of the best studied variations of drug metabolism. In this case, the so-called "poor metabolizer" (hereinafter referred to as PM) phenotype is inherited as an autosomal-recessive trait and occurs with a frequency of 5-10% in the European and North American population (Meyer et al., Advances in Drug Research 19, 197-241 [1989] and Eichelbaum, ISI Atlas of Science; 243-251 [1988]). Phenotype means a physical or behavioral trait of an organism. In the case of debrisoquine polymorphism, the PM-phenotype is associated with the inability of efficient metabolization of over 25 drugs, including antiarrhythmics (e.g. flecainide and propafenone), antidepressants (e.g. imipramine, nortriptyline, clomipramine), neuroleptics (e.g. perphenazine and thioridazine), antianginals (perhexiline) and opioids (e.g. dextromethorphan and codeine (Meyer et al., Pharmac. Ther. 46, 297-308 [1990]). People who suffer from this deficiency of drug metabolism often experience exaggerated pharmacological or toxic responses when they are treated with usual doses of drugs.
In addition to the debrisoquine polymorphism, two other genetic polymorphisms of drug metabolism have been studied at the molecular level. These are the mephenytoin polymorphism which is located within the P450IIC subfamily and the acetylation polymorphism (Meyer et al., Advances in Drug Research 19, 197-241 [1989]). The reasons for these metabolic disorders seem to be the same as for the debrisoquine polymorphism in that distinct mutations in corresponding genes coding for the metabolizing enzymes do exist.
Previous studies have revealed that the debrisoquine PM phenotype is caused by the absence in the liver of a specific cytochrome P450 isozyme, designated P450IID6 (Nebert et al., DNA 8, 1-13 [1989]) or P450dbl (Zanger et al., Biochemistry 27, 5447-5454 [1988]). The gene for P450IID6, designated CYP2D6 (Nebert et al., supra), has been localized to chromosome 22 (Gonzalez et al., Genomics 2, 174-179 [1988]). A presumed pseudogene CYP2D7 and a definite pseudogene CYP2D8 are localized 5' of the CYP2D6 locus (FIG. 1), (Kimura et al., Am. J. Hum. Gem 45, 889-904 [1989]). Aberrant splicing of its premRNA was observed in livers of PMs and could explain its absence (Gonzalez et al., Nature 331,442-446 [1988]). These publications by Gonzalez et al. or Kimura et al. mentioned above describe cDNA's (and not genomic DNA sequences) which do not define specific mutations of the CYP2D6 gene which would allow the determination of the genotype and the assignment of the corresponding debrisoquine phenotype. In further studies, using restriction fragment length polymorphism (RFLP) analysis of leukocyte DNA, several mutant alleles of the P450IID6 gene locus (CYP2D) associated with the PM-phenotype were identified (Skoda et al., Proc. Natl. Acad. Sci. USA 85, 5240-5243 [1988]). After digestion of genomic DNA with the restriction enzyme XbaI these two alleles produced characteristic fragments of 11.5 kb and 44 kb respectively (FIG. 2). However, only the genotypes 44/44 kb, 44/11.5 kb or 11.5/11.5 kb so far predicted the PM-phenotype. It had been hoped that RFLP analysis would allow genotyping of all the PMs. In practice only 25% of PMs could be predicted after tests with numerous restriction endonucleases. All the extensive metabolizers (EM) and the remainder of PMs (75%) had one or two 29 kb fragments which can represent both an active (wild-type) or defective allele. Therefore, the RFLP-patterns have the disadvantage of being noninformative in regard to phenotype. To account for all mutant alleles, those represented by XbaI-29kb fragments therefore need further genomic characterization.
Previously the phenotype was determined by the administration of a test drug (debrisoquine, sparteine or dextrometorphan) followed by collection of urine for several hours and determination of the ratio between parent drug and its metabolite (urinary metabolic ratio). This procedure has considerable limitations because of adverse drug reactions, drug interactions and the confounding effect of diseases. Identification of the mutant genes causing the PM phenotype (i.e. the genotype) followed by the development of tests for the detection of the respective genotype, is therefore desired.
The acetylation polymorphism is also a classical example of a genetic defect in drug metabolism. It was observed over a quarter of a century ago with the advent of isoniazid therapy for tuberculosis by Hughes et al. (Am. Rev. Respir. Dis., 70, 266-273 [19541]). Patients could be classified as "rapid" ("fast") or "slow" eliminators of isoniazid and family studies revealed that the ability to eliminate isoniazid was determined by two alleles at a single autosomal gene locus, slow acetylators being homozygous for a recessive allele as described by Evans et al. in Br. Med. Jr., 2, 485-491 (1960). The polymorphism of N-acetylation has recently been reviewed by Meyer et al. in Advances in Drug Research 19, 197-241 (1989).
The proportions of rapid (RA) and slow acetylators (SA) vary remarkably in different ethnic and/or geographic populations. For example, the percentage of slow acetylators among Canadian Eskimos is 5%, whereas it rises to 83% among Egyptians and 90% among Moroccans. Most populations in Europe and North America have an approximately equal number of rapid and slow acetylators.
Numerous subsequent studies have demonstrated that the acetylation polymorphism affects the metabolism of a wide variety of other arylamine and hydrazine drugs and numerous foreign chemicals. These include the drugs sulfamethazine (SMZ) and several other sulfonamides, hydralazine, procainamide, dapsone, p-aminobenzoic acid (PABA), phenelzine and aminoglutethimide. The polymorphism also involves the metabolism of caffeine, clonazepam and nitrazepam as well as the potential arylamine carcinogens benzidine, 2-aminoflorene and .beta.-naphthylamine.
The phenotyping procedures in case of the acetylation polymorphism using isonazid were later replaced by testing with sulfamethazine or dapsone. More recently, a phenotyping procedure using caffeine as a test substance has been developed and described by Grant et al. in Brit. J. Clin. Pharmacol. 17, 459-464 (1984) and further refined by Tang et al. as described in Clin. Pharmacol. Ther. 42, 509-513 (1987). These procedures have the same practical limitations as described above for the debrisoquine polymorphism. A more practical and unambiguous procedure for the determination of this polymorphism is therefore desired.
Recently the gene coding for the human hepatic arylamine N-acetyltransferase (NAT), which is responsible for the acetylation polymorphism described above, has been cloned from human leukocyte DNA by Blum et al. (DNA and Cell Biology 9, 193-203 The sequences of these genes have the following designations and accession numbers in the EMBL data library: NAT1, hgnat-a, X17059; NAT2, hgnat-b, X14672. Two genes, designated NAT1 and NAT2, have been assigned to human chromosome 8, pter-q11. The product of the NAT2 gene had an identical apparent molecular weight as the NAT protein detected in human liver cytosol. The two human genes, NAT1 and NAT2, encoding two NAT proteins were cloned and characterized. The numbers given to the base sequences of NAT1 and NAT2 were also used in this specification. NAT2 was identified as the gene encoding the polymorphic NAT2 isozyme. Knowing the mutations of the allelic variants of the NAT2 gene would allow the development of methods for the detection of the genotype instead of the PM phenotype.